Dmi6000 b inverted fluorescent microscope

HEK293-Flp-In cells stably transfected by our Double-Flp-In Method with NTCP-CFP and NTCP-YFP, a classical Foerster Resonance Energy Transfer (FRET) pair, were seeded onto IBIDI chamber-slides to reach confluency at the day of microscopy. For comparison HEK293-MSR cells were seeded like above and were transiently transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Darmstadt, Germany) with an equimolar number of premixed plasmids of pcDNA5 vectors coding for NTCP-CFP or NTCP-YFP both under the control of the identical CMV-promoter, which is also applied by the Double-Flp-In Method. After 48 h of standard incubation, slides were washed twice with PBS and transferred to microscopy at room temperature covered in PBS. Images were taken with a Leica DMI6000 B inverted fluorescent microscope at 40× objective magnification. For qualitative analysis and comparison of expression levels and patterns of the fluorescent proteins CFP and YFP channels were adjusted to yield similar signal intensities. Phase contrast channel was applied to demonstrate the confluency of the cell layer and transfection rates. Staining of the cell nuclei and the fixation of the cells was deliberately avoided to not interfere with CFP and YFP signals.

OE-129WT cells and OE-TLR3(-) cells were grown to confluence in 96-well Ibidi plates (Ibidi USA; Fitchburg, Wisconsin) before being either mock-infected or infected with 1 IFU/ cell C. muridarum at 37°C for up to 36hrs. At the specified time-point, the cells were carefully rinsed with PBS before being fixed 10 min at room temperature using 10% neutral-buffered formalin (Sigma). The monolayers were washed at room temperature 3 times for 5 min using PBS. After washing in PBS, the cells were blocked and permeabilized for 30 min using PBS containing 1% BSA and 0.1% saponin. After permeabilization, cells were washed in blocking buffer (PBS + 1% BSA) at room temperature for 5 min, and the primary antibody (either Claudin-1; 1:50 dilution, ZO-1; 1:100 dilution, or JAM-1; 1:200 dilution) was added to the cells for 1hr at room temperature. After washing at room temperature 3 times for 5 min using PBS, the cells were then incubated in the dark at room temperature for 1hr using either goat anti-rabbit, goat anti-mouse, or donkey anti-goat 2° antibody conjugated with Alexa Fluor-594 (1:1000 dilution, Molecular Probes). Finally, the cells were washed 3 times for 5 min using PBS and counterstained with DAPI to identify the nuclear DNA. Duplicates processed without primary antibodies served as negative controls. Fluorescence was imaged using a Leica DMI 6000B inverted fluorescent microscope.

Each slide was incubated 2x in BioGenex brand EZ-Dewax solution for 5 min each incubation and then washed 3x with distilled water. Sections were blocked with 10% FCS, 0.1% Triton X-100 in TBS-T for 1 hr. at room temp. Typically, sections were incubated in primary antibody using 2ml per slide at a 1:200 dilution in blocking buffer for 1 hr. at room temp and were then washed 3x with PBS for 5 min each wash. Next, sections were incubated in a secondary fluorescently-labeled antibody using 2ml per slide at a 1:200 dilution in blocking buffer for 1 hr. at room temp and were then also washed 3x with PBS for 5 min each wash. For reference, sections were stained with 1:2000 DAPI for 5 min. at room temp to visualize nuclei in regards to other cellular components of interest and were rinsed 1x with PBS and then were mounted using VectaShield Mounting Media. Immunofluorescence microscopy was performed using a Leica DMI 6000B inverted fluorescent microscope.

Eyes were marked dorsally via cauterization, and 12 μm sections were prepared as previously described [117 (link)]. Sections were blocked either in blocking solution (PB-Salt (0.1 M phosphate buffer with 0.8% NaCl and 0.02% KCl) with 0.3% Triton-X (Sigma-Aldrich) and 3% goat serum (Sigma-Aldrich) or 2% horse serum (Sigma-Aldrich)), followed by primary antibody incubation overnight at 4 °C. After three washes with PB-Salt, secondary antibody incubation was performed for an hour at room temperature. Nuclear counterstaining was performed with 4′,6-diamidine-2′-phenylindole di-hydrochloride (DAPI, Thermo Fisher Scientific). Sections were mounted using Mowiol (Sigma-Aldrich) and imaged with a Leica DMI6000B inverted fluorescent microscope (Leica Microsystems, Wetzlar, Germany). Primary and secondary antibodies used in the paper are listed in Additional file 16. Z-stacks were merged with max projection in ImageJ. Images were optimized for histogram minimum and maximum values using Adobe Photoshop.